The level of cholesterol in blood is a significant indicator of risk of coronary heart disease. “Total cholesterol” includes low density lipoproteins (LDL), very low density lipoproteins (VLDL) and high density lipoproteins (HDL). It is well established from epidemiological and clinical studies that there is a positive correlation between levels of LDL and VLDL cholesterol (“bad” cholesterol) and coronary heart disease and a negative correlation between levels of HDL cholesterol (“good” cholesterol) and coronary heart disease. The level of total cholesterol in blood, which is a measure of the sum total of HDL, LDL, VLDL and chylomicrons, is not generally regarded as an adequate indicator of the risk of coronary heart disease because the overall level of total cholesterol does not reveal the relative proportions of HDL, LDL and VLDL. To better assess the risk of heart disease, it is desirable to determine the amount of HDL cholesterol in addition to total cholesterol.
However, to measure HDL separately, two significant treatment steps to a whole blood sample are usually necessary. First, blood cells (especially erythrocytes) interfere with typical colorimetric tests and therefore must be separated from the whole blood sample to produce plasma or serum. Second, non-HDLs (i.e., LDL, VLDL and chylomicrons) must be removed from the plasma to be tested because reagents used to determine the level of HDL will also react with LDL and VLDL.
The conventional method of removing blood cells from whole blood is centrifugation. Centrifugation is a process step requiring time and a centrifuge, and it is therefore unacceptable for blood tests that are conducted in many physicians' offices, on-site testing by medical technicians, and testing by patients at home. Further, centrifugation can cause problems with separating supernatant and blood cake.
A significant advance to the field of diagnostic devices was ushered in with the discovery by Vogel, et al. (U.S. Pat. No. 4,477,575) in the early 1980's that glass fibers could be used to separate red cells from whole blood. Because of optical and chemical interference from hemoglobin in red cells, the only material that could be measured in whole blood at that time was glucose, using early test strips that required the red cells to be washed or wiped off after glucose had permeated a paper-based matrix (for example, U.S. Pat. No. 3,298,789 to Mast). Glass fibers separate red blood cells by physical and chemical adhesion of the cell surface to the glass fibers. Even today, however, the precise nature of the attraction between glass fibers and red blood cells is not clearly understood. Weak chemical bonding, van der Waals forces, hydrogen bonding or other intermolecular forces may have a role in this attraction.
The discovery that glass fibers separate blood cells, however, allowed, for the first time, measurement of cholesterol and other blood components in a doctor's office instead of a reference laboratory, and the first commercial device to utilize this technology was Boehringer Mannheim's (now Roche Diagnostics) Reflotron® instrument. This advance was subsequently incorporated into test strips, allowing blood testing at home.
Notwithstanding the significant achievement of the '575 patent, applicants have found that commercially available test strips embodying the '575 patent and its progeny are “lateral flow devices.” The defining feature of a lateral flow device is the presence of a sample application point that is laterally offset (along the axis of the test strip) from the sample reading area of the test strip. For example, certain commercially available devices that appear to embody the teachings of the '575 patent include a blood application area at one end of the elongated test strip and a test reading area at the other end. A whole blood sample is deposited at one end of the glass fiber blood separation layer, and plasma migrates to the other end at a greater rate than do red blood cells. However, it has been experimentally determined by applicants that red blood cells from the sample that is placed on the disclosed glass fiber matrix eventually migrate tangentially across the fiber matrix, albeit at a slower rate than plasma. Further, some hemolysis of the erythrocytes eventually occurs in the glass fiber layer.
Furthermore, applicants have found that some commercially available total cholesterol test strips are configured such that the reaction layer is not initially in contact with the glass fiber blood separation layer. Instead, the reaction layer is not brought into fluid-conveying contact with the glass fiber layer until the glass fiber layer is filled with plasma. This happens at a predetermined time after an adequate amount of plasma, but not red blood cells, has migrated laterally to a designated location on the glass fiber layer. Timing is thus important to the successful use of such test strips. If the reaction layer is brought into contact with the glass fiber layer too soon after depositing the blood sample on the strip, not enough plasma will have migrated to the designated area of the strip and the analyte concentration determined may be inaccurately low. On the other hand, if the reaction layer and glass fiber layer are not brought into contact soon enough, hemolyzed and intact red blood cells will migrate to the test area and interfere with the color to be measured from the reaction. Applicants have found these commercially available test strips to be highly accurate when used as directed. However, it would be desirable to avoid the process step of bringing the test layer into contact with the blood separation layer.
Another blood separation scheme is disclosed in U.S. Pat. No. 5,135,716 (Thakore) and the abandoned application from which it claims priority. The device described in the '716 patent is also a lateral flow device but operates differently than the glass fiber matrices described in the '575 patent. The '716 device purports to employ an industrial “cross-flow” or “tangential filtration” technique on a miniature scale. The blood sample is applied to one end of a physical transport medium and is moved laterally thereby, along the underside of a microporous plasma separation membrane. Blood is separated at the bottom surface of this microporous plasma separation membrane, and clean plasma is obtained on the top side of the membrane. The transport medium provides the driving force for lateral movement of blood, such that blood is swept across the underside of the microporous plasma separation membrane, thereby cleaning it and preventing it from clogging with red blood cells. However, to Applicants' knowledge, there has never been a commercial test strip produced or sold under the '716 patent, likely because the blood separation technology described in the patent, among other things, is simply unworkable.
Another alternate approach to centrifugation to separate blood cells is disclosed in U.S. Pat. No. 5,876,605 (Kitajima et al.). The method involves mixing an aqueous solution of an inorganic salt or an amino acid or salt thereof with whole blood in an amount 20% or less of the whole blood volume and then filtering the whole blood to remove blood cell components. While satisfactory results are apparently achieved with the wet chemistry method disclosed, the '605 patent teaches that the technique cannot be successfully adapted to dry test layers such as glass fiber matrices. '605 patent, column 11, lines 1–30.
Test strips for precipitation and separation of non-HDL cholesterol from HDL cholesterols in a plasma sample are disclosed by U.S. Pat. No. 5,426,030 (Rittersdorf et al.) and its progeny. This separation technology involves a test strip with two layers in contact with one another. The first layer is made from glass fibers in the form of fleeces, the glass fibers having a diameter from 3 to 100 μm. The first layer is hydrophilic, having a thickness between 20–250 μm and pore sizes between 0.2–20 μm, and is impregnated with a precipitating agent that precipitates non-HDLs but not HDLs. The second layer is preferably a mesh glass fiber layer with fibers of a diameter of 0.2 to 10.0 μm. Precipitation of non-HDL cholesterols occurs in the first layer and separation of the non-HDL precipitants from the plasma occurs in the second layer.
U.S. Pat. No. 5,135,716 (Thakore), discussed above, discloses a multilayer strip, two of such layers being used for precipitating and then separating non-HDLs from plasma, respectively. The '716 patent also suggests that precipitation and separation of non-HDLs from plasma can be carried out in a single “asymmetric” carrier layer. The asymmetric layer essentially operates as two layers, in that the top portion of the layer includes large pores to allow fluid movement and precipitation, whereas the bottom portion of the layer includes smaller pores to trap the precipitants. Applicants have found that this disclosure does not rise beyond mere speculation, in that no examples or enabling disclosure of the single asymmetric layer technology to separate non-HDLs from plasma are found in the '716 patent.
Yet another elaborate device to measure the concentration of HDL cholesterol from a whole blood sample is disclosed in U.S. Pat. No. 5,213,965 (Jones) and other related and commonly assigned patents. The device includes a well in which the whole blood sample is deposited and then drawn through a capillary to a sieving pad made of fibrous material. The sieving pad achieves initial separation of blood cells from plasma on the basis of the blood cell's slower migration rate therethrough. The sieving pad is covered with a microporous membrane which further filters blood cells. Covering the microporous membrane is a reagent reservoir membrane containing precipitating agents for non-HDLs. On top of and extending laterally beyond the reagent reservoir is an elongate matrix which distributes the sample laterally after it leaves the reservoir. Finally, one or more test pads are positioned above and biased apart from the elongate matrix. Plasma exits the filtering membrane and enters the reagent reservoir where non-HDLs are precipitated. The plasma and non-HDL precipitates then flow from the reservoir and migrate laterally through the elongate matrix.
Undesirably, the device disclosed by the '965 patent relies upon not one, but two, separate chromatographic operations, the first being blood separation in the sieving pad, and the second being separation of non-HDLs across the elongate matrix. Proper timing is crucial to these chromatographic operations. Further, the device disclosed by the '965 patent is undesirably complex. For example, it requires a well, a capillary tube, two layers to separate blood cells, and two layers to precipitate and then separate non-HDLs. Finally, the test pads must be kept spaced apart from the elongate matrix until the entire operation is properly timed, whereupon the test plate having the test pads thereon can be depressed against the elongate matrix. Of course, depressing the test pad creates yet another undesirable process step and introduces further potential for error.
U.S. Pat. No. 5,460,974 (Kozak et al.) discloses a test device for measuring HDL cholesterol. The device relies upon a blood separation layer having incorporated therein about 25 to about 250 units of an agglutinin, about 50 to about 150 NIH units of a coagulant or a mixture thereof to agglutinize or coagulate the cellular components of the undiluted whole blood sample. The plasma is then passed into an adjacent layer by gravity to separate the LDL and VLDL fractions from the plasma, followed by a layer which filters the non-HDLs. Applicants have found that using an agglutinin or a coagulant to separate blood cells is undesirable because it affects the measured test result.
It is desirable to avoid the lateral flow schemes, chromatographic operations, complex devices and the timing operations that are required for blood cell separation in the patents discussed above. It would also be desirable to achieve a blood separation mechanism that is more efficient and dependable than those listed above. It is also desirable to simplify non-HDL separation from plasma. Generally, it is desirable to provide a test strip for measuring concentration of HDL cholesterol that is more reliable, economical and easier to use than the prior art devices discussed above.